Biomolecules such as proteins, antibodies, DNA strands, red blood cells and semen, molecular and biological moieties, and large molecules in general, are commonly detected and separated using electrophoresis in gels and other media. Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Ferdinand Frederic Reuss (Moscow State University), who noticed that the application of a constant electric field caused clay particles dispersed in water to migrate. Electrophoresis is ultimately caused by the presence of a charged interface between the particle surface and the surrounding fluid, and it is the basis for a number of analytical techniques used in biochemistry for separating molecules by size, charge or binding affinity.
Electrophoresis is a technique used in laboratories to separate macromolecules based on their size. The technique involves applying a negative charge so that particles such as proteins move toward a positive charge. This technique is used for both DNA and RNA analysis. Polyacrylamide gel electrophoresis (PAGE) has a clearer resolution than electrophoresis based in agarose and is more suitable for quantitative analysis. Using PAGE, DNA foot-printing can identify how proteins bind to DNA. PAGE can be used to separate proteins by size, density and purity, and further may be used for plasmid analysis for developing an understanding of bacteria becoming resistant to antibiotics.
Recently, dielectrophoresis (DEP), which uses electric field gradients, has been utilized for similar applications and cell separation. DEP does not require that the macromolecules be charged, and instead relies on the polarizability of the macromolecules. Dielectrophoresis occurs when a polarizable particle is suspended in a non-uniform electric field. The electric field polarizes the particles, and the particles' poles experience a force along the field lines, which force can be either attractive or repulsive, according to the orientation of the dipole. Since the field is non-uniform, the pole experiencing the greater electric field will dominate the other, and the particle will move. The orientation of the dipole is dependent on the relative polarizability of the particle and medium, in accordance with Maxwell-Wagner-Sillars polarization. Further, since the direction of the force is dependent on field gradient rather than field direction, dielectrophoresis will occur in alternating current as well as direct current electric fields; polarization, and hence the direction of the force, will depend on the relative polarizabilities of particle and medium. If the particle moves in the direction of increasing electric field, the behavior is referred to as positive DEP. If acting to move the particle away from high field regions, it is known as negative DEP (nDEP). As the relative polarizabilities of the particle and medium are frequency dependent, varying the energizing signal and measuring the manner in which the force changes can be used to determine the electrical properties of particles; this allows for the elimination of electrophoretic motion of particles due to inherent particle charge.
Additional phenomena associated with dielectrophoresis are electrorotation and traveling wave dielectrophoresis (TWDEP). These require complex signal generation equipment and patterned electrode structures to create the required rotating or traveling electric fields; as a result of this complexity, these techniques have found less favor than conventional dielectrophoresis among researchers.
In addition to electrophoretic separation, identification and separation is accomplished by methods of attaching proteins or molecule-specific fluorescent or chemiluminescent markers used in the Southern Blot and Western Blot assays to identify electrophoretically separated macromolecules obtained from the lycing of cells. The techniques of electrophoresis and blotting are capable of handling biological moieties ranging from 10-1000 kD, or 3-100 nanometers along various dimensions. While electrophoresis and blotting technologies are expected to constitute a $2 billion market by 2020, there are several shortcomings associated with these technologies, including a one-day performance cycle duration, being limited to charged species, difficulty handling large molecules (e.g., titin), a requirement of milliliter samples and several reagents, and an inability to produce information regarding dielectric properties. More recently, nonlinear four-wave mixing techniques have been employed to identify specific molecules in conjunction with electrophoretic or dielectrophoretic separation. For example, ultrasensitive detection of proteins and antibodies by absorption-based laser wave-mixing detection using a chromophore label has been demonstrated by Tong et al. The four-wave mixing signal results in an absorption grating formed by the linkage of a non-fluorescing chromophore label, Coomassie Brilliant Blue (CBB), which absorbs the laser radiation.
Moreover, dynamic light scattering (DLS) is another approach for determining the size distribution profile of particles in suspension or polymers in solution. See Lim et al., Nanoscale Research Letters 8:381 (2013). This technology is expected to constitute a $200 million market by 2020, but suffers many of the same problems as electrophoresis and blotting, with shortcomings including a requirement of microliter samples, being limited by low level signals, and an inability to produce information regarding dielectric properties. Further, this technology produces large errors outside a narrow size range.
In light of these deficiencies associated with electrophoresis/blotting and DLS, particularly the requirement for large amounts of material in order to perform these processes as well as the large errors, e.g., up to 500%, produced based on concentration, there exists a need for superior means of identifying and separating macromolecules.